Screening And Identification Of Salt-tolerant Mutants Of Apple Rootstock

Apple is one of the most important fruit crops in the world by graft breeding, so rootstock was the key of apple's salt-tolerant ability. Screening and breeding salt tolerant mutants could improve apple salt-tolerant ability. Recently tissue culture and mutagenesis technique were used in screening salt tolerant mutant successfully in many crops. But the mutant's reality was often doubted, because the mutant was likely to the adaptability to environment stress, but not the change of genotype. So it is the most important to identify the genotype of the mutants.The salt-tolerant mutant selection and identification was systematically studied by using NaCl as selection stress with 78-48 and EH-78-48 by irradiation and tissue culture. The main results were as follows:1. Leaf explant regeneration systems of 78-48 and EH-78-48 were established. The experiment results indicated that the optimum medium compositions of apple were MS + BA2.0mg ? L^-1 + NAA0.1 mg ? L^-1) or MS + TDZ1.0mg ? L^-1 + NAA0.1 mg ? L^-1. The optimum estoping light time was 20 days.2. The growth and leaf regeneration of 78-48 and EH-78-48 were distinctly restainer66d by ⁶⁰Co-γ irradiation, the proportion of abnormality leaf was inhanced.3. The obvious mutation leaves were used to regeneration. To obtain salt-tolerant mutant, high-level NaCl (1.0% & 1.2%) was added into the culture medium directly. Though salt stress culture for many successive generations, the salt tolerant mutants L line and K were obtained.4. The shape index was observed of the mutants and control. The results indicated that the survival rate of their mutants cultured on salt medium were higher than control, but the injury rate and injury index of mutants were lower than control. The mutants showed higher level of salt tolerance.5. The physiological and biological indexs were studied to the salt-tolerant mutants. The result indicated that the cell membrane permeability was increased, MDA, O₂^- and Cl^- contents were improved in apple rootstock under the salt stressand were higher than that of control. SOD and POD activity were increased and were higher than that of control. Na+ content of the mutant was inhanced and K+ content was decreased under salt stress, Na+/K+ rate was increade and was lower than that of control. The results showed that the mutants were bear to salt stress.6. The content of Pas, Spm, Spd and Put was first increased and decreased under the condition of salt stress. The ratio of (Spd+Spm) /Put was increased with the salt concentration increasing. The content of Pas, Spm, Spd and Put and the ratio of (Spd+Spm) /Put of salt-tolerant mutants were higher significantly than that of control.7. Under the condition of salt stress, the content of IAA, GA, ZR and the ratio of ZR/ABA were dereased gradually, but the content of ABA was increased. The ABA concent of salt-tolerant mutants was higher significantly than that of control.8. The correlation of physiological and biological indexs showed that the contents of MDA, O2", Cl", SOD activity and Pro was positive to the cell membrane permeability. The content of ABA, POD and Pas was also positive to the cell membrane permeability, but the coefficient correlation was small, so we thought that the cell membrane permeability, the content ofO2", Cl" and SOD activity were used to salt tolerant identification.9. Through RAPD analysis of 78-48 and its mutants, the results showed that 32 out of 36 randomized primers had DNA polymorphism amplifided and 546 bits were detected in apple rootstocks genome. The 40 and 38 DNA polymorphism bits were detected to two mutated lines Li, L2 of 78-48 defferently, 37 DNA polymorphism bits were detected to mutated lines K2 of EH-78-48 definitely. Results showed the mutants varied at the DNA molecular level, which provided an evidence of the mutation.10. The DNA polymorphism fragments were cloned and sequenced. The results indicated that the randomized primer S174 and S373 were possible salt marker and linked to the salt tolerant mutation locus. Compared with control, the mutants of EH-78-48, L2 and K2 were detected to have the mutation in DNA level and the mutation was correlation to the salt-tolerant. The mutant Li was salt tolerant in physiological and biological and shape indexs, and were detected to have the mutation in DNA level. The result of cloning sequencing showed that the mutation was not correlation to salt tolerant, this indicated that the mutation was possiblecorrelation to other character or the quantity of primers was small, whether or not the mutation of Li was related to salt tolerant was need to be confirmed.11. Through AFLP analysis of rootstock 78-48 and its mutants, two pairs out of ten pairs primers distiguished the difference of the mutants and control. Two pairs primers had 118 bands amplified and 118 bits were detected. Having polymorphism amplified indicated that the mutants varied at the DNA molecular level. Results showed that 68 bits were detected in the genome, was 57.63% in the all polymorphism bands. This showed that 57.63% polymorphism bits exsisted the difference in the materials. AFLP technology was used to identify the mutants and the efficiency was high.